Heterologous Expression and Characterization of a Thermostable α-L-Rhamnosidase from Thermoclostridium stercorarium subsp. thermolacticum DSM 2910 and Its Application in the Biotransformation of Rutin

An α-L-rhamnosidase gene from Thermoclostridium. stercorarium subsp. thermolacticum DSM 2910 (TstRhaA) was cloned and expressed. The maximum TstRhaA activity of the protein reached 25.2 U/ml, and the molecular mass was approximately 106.6 kDa. The protein was purified 8.0-fold by Ni-TED affinity with an overall recovery of 16.6% and a specific activity of 187.9 U/mg. TstRhaA activity was the highest at 65°C and pH 6.5. In addition, it exhibited excellent thermal stability, better pH stability, good tolerance to low concentrations of organic reagents, and high catalytic activity for p-nitrophenyl-α-L-rhamnopyranoside (pNPR). Substrate specificity studies showed that TstRhaA exhibited a high specific activity for rutin. At 60°C, pH 6.5, and 0.3 U/ml enzyme dosage, 60 g/l rutin was converted to 45.55 g/l isoquercitrin within 150 min. The molar conversion rate of rutin and the yield of isoquercitrin were 99.8% and 12.22 g/l/h, respectively. The results suggested that TstRhaA could be used for mass production of isoquercitrin.


Introduction
Isoquercitrin is a rare flavonol glycoside that exhibits anti-cancer, anti-influenza, antioxidant, anti-allergic, antihypertensive and other pharmacological activities [1][2][3][4][5], and is attracting increasing attention.Isoquercitrin is also a key synthetic intermediate for the preparation of enzymatically modified isoquercitrin, which is the only flavonoid food additive approved by the US FDA, and the GRAS Notice for EMIQ is GRN 000220 [6,7].However, extracting and isolating isoquercitrin from plants on a large scale is difficult because the content of isoquercitrin in nature is low, greatly restricting its large-scale application [8].Rutin is abundant in many plants [9], and isoquercitrin differs from rutin by only one rhamnose residue.Therefore, concerting multicomponent rutin into the rare component isoquercitrin is a good strategy [10][11][12][13].
α-L-Rhamnosidase (E.C. 3.2.1.40)is ubiquitous in natural sources, such as fungi, mammalian tissues, plants, and bacteria [14][15][16].It can efficiently and specifically cleave a variety of natural compounds containing nonreducing terminal L-rhamnose residues, such as hydrolyzed rutin as isoquercitrin and icaritin C as icariin [17,18].In addition, a few α-L-rhamnosidases can catalyze the reverse hydrolytic synthesis of rhamnosides using rhamnose as a donor, such as the rhamnosylation of mannitol and the rhamnosylation of phenolic compounds [19,20].α-L-rhamnosidase belongs to the GH78, GH13, and GH106 glycoside hydrolase families [21], among which the GH78 family is the majority.This enzyme has many applications in industry [22].However, most reported α-L-rhamnosidase is not very thermostable at medium and high temperatures, which greatly limits its industrial application.
The CSTERTH_05025 protein of the GH78 family from Thermoclostridium stercorarium subsp.thermolacticum DSM 2910 was listed in the Carbohydrate-Active enZYmes (CAZy) database.We speculated that the enzyme should exhibit α-L-rhamnosidase activity and that the enzyme might be more thermostable.Therefore, in our An α-L-rhamnosidase gene from Thermoclostridium.stercorarium subsp.thermolacticum DSM 2910 (TstRhaA) was cloned and expressed.The maximum TstRhaA activity of the protein reached 25.2 U/ml, and the molecular mass was approximately 106.6 kDa.The protein was purified 8.0-fold by Ni-TED affinity with an overall recovery of 16.6% and a specific activity of 187.9 U/mg.TstRhaA activity was the highest at 65°C and pH 6.5.In addition, it exhibited excellent thermal stability, better pH stability, good tolerance to low concentrations of organic reagents, and high catalytic activity for pnitrophenyl-α-L-rhamnopyranoside (pNPR).Substrate specificity studies showed that TstRhaA exhibited a high specific activity for rutin.At 60°C, pH 6.5, and 0.3 U/ml enzyme dosage, 60 g/l rutin was converted to 45.55 g/l isoquercitrin within 150 min.The molar conversion rate of rutin and the yield of isoquercitrin were 99.8% and 12.22 g/l/h, respectively.The results suggested that TstRhaA could be used for mass production of isoquercitrin.
study, the CSTERTH_05025 protein gene from Thermoclostridium stercorarium subsp.thermolacticum DSM 2910 was cloned, expressed and characterized.The results showed that the enzyme exhibits α-L-rhamnosidase activity and good thermal stability.Moreover, the conditions for the conversion of rutin to isoquercitrin were optimized, and the results showed that the enzyme exhibits high selectivity for the transformation of rutin to isoquercitrin.Our results suggested that TstRhaA shows great potential for hydrolyzing rutin to prepare isoquercitrin.
The DNA purification kit, Plasmid MiniPrep Kit, DNA Marker, restriction endonucleases NcoI, XhoI, and PCR SuperMix were purchased from TransGen Biotechnology (China).Artificial substrates of the pNP series, hesperidin, and naringin were obtained from Sigma-Aldrich (USA).Rutin and myricetrin were purchased from Chendu Must Bio-Technology (China).The Ni-TED affinity column, T4 DNA ligase, and modified Bradford protein assay kit were obtained from Sangon Biotech (China).

Plasmid and Recombinant Bacteria Constructions
The TstRhaA gene was amplified from T. stercorarium DSM 2910 genomic DNA using PCR SuperMix by PCR.Primers TstRhaA-F (CATG CCATGG AAATGATGAGAGTTTA TAAC) and TstRhaA -R (CCG CTCGAG TTCCACAGTAACTTTGCTGAG) were used for gene amplification.The underlined bases are restriction enzyme sites.The PCR product was cleaved using the restriction enzymes NcoI and XhoI and then inserted into pET-20b (+) to generate the expression plasmid pET-TstRhaA.Finally, the positive expression plasmid pET-TstRhaA was transformed into E. coli BL21(DE3) and then screened by ampicillin resistance.

Sequence Analysis of TstRhaA
Potential ORFs of TstRhaA were found using the CAZy database.The National Center for Biotechnology Information (NCBI) database was used for BLAST sequence alignment.Several GH78 family α-L-rhamnosidases of different origins were used for sequence alignment.Multiple protein sequence alignment was performed using Clustal X1.9.Molecular weight and theoretical pI were predicted using DNAMAN software.

Optimization of TstRhaA Culture Conditions
The positive transformants were inoculated in 5 ml LB medium containing ampicillin at a final concentration of 100 mg/l and incubated for 16 h at 37°C and 180 r/min.The cells were then inoculated into 150 ml LB medium containing ampicillin at a final concentration of 100 mg/l according to the 1% inoculum to continue the culture.

Methods for Purifying TstRhaA
At the end of incubation, the cells were collected by centrifugation, resuspended in 20 mM Tris-HCl buffer (pH 7.9) containing 5 mM imidazole, and finally crushed by a pressure crusher.The resulting supernatant was purified using a Ni-TED affinity column, and the purified TstRhaA was eluted with 20 mM Tris-HCl buffer (pH 7.9) containing 1 M imidazole.The expressed proteins were detected by SDS-PAGE and analyzed by a gel imager.The protein concentration was assayed using a Bradford protein assay kit with bovine serum albumin as the standard [23].

Methods for Measuring Enzyme Activity
TstRhaA activity assays were performed using pNPR as a substrate in 100 μl of reaction solution with 1 mM pNPR at 65 o C and 100 mM pH 6.5 citrate-phosphate buffer.The reaction was terminated by adding 300 μl of 1 M sodium carbonate after 5 min of reaction.The released p-nitrophenol (pNP) was then measured at 405 nm.One unit of TstRhaA activity was defined as the amount of enzyme liberating 1 micromole of pNP per minute at 65 o C and pH 6.5.

Determination of Enzymatic Properties
pNPR was used to characterize the enzymatic properties of TstRhaA.The optimum pH of TstRhaA was determined by measuring the enzyme activity from pH 5.0 to 8.0.The optimum temperature of TstRhaA was determined by measuring the enzyme activity from 35 to 75 o C.
The pH stability of TstRhaA was assessed by measuring the remaining enzyme activity after TstRhaA was incubated for 4 h at 4 o C, 45 o C, and 60 o C in a buffer pH range of 5.0 to 8.0.The thermal stability of TstRhaA was determined by measuring the remaining enzyme activity after TstRhaA was incubated for different times at 60 o C and 65 o C in pH 6.5 buffer.
The organic solvent tolerance of TstRhaA was determined by adding methanol, ethanol, or DMSO to the reaction mixture at final concentrations of 0%, 10%, 15%, and 20%, respectively.
The substrate specificity of the enzyme was tested by using the synthetic substrates pNPR, pNPGlu, pNPArf, pNPArp, pNPXyl, pNPGal and plant-derived flavonoid compounds rutin, myricetrin, hesperidin, and naringin.The specific enzymatic activity of TstRhaA against different substrates was determined in the reaction system at a substrate concentration of 1.0 mM.Different pNPR concentrations (0.1, 0.2, 0.4, 0.6, 0.8, and 1 mM) were measured at pH 6.5 and 65 o C to determine the initial rate TstRhaA kinetic constants.

Analysis of Rutin Degradation
The reaction system was 200 μl, which contained rutin, buffer, and TstRhaA.The effects of different pH values, temperature, enzyme amounts and reaction times on the conversion rate of hydrolyzed rutin were studied.The hydrolysis of rutin by TstRhaA was investigated by HPLC.The HPLC method was the same as we previously performed [24].The rutin transformation rate and isoquercitrin generation yield were calculated as follows.

Statistical Analysis
All experiments were repeated three times.Data represent the means ± SDs of three replicates.Tukey's test was used at 95% confidence intervals (p <0.05).

Analyzing the Sequence of TstRhaA
The full-length α-L-rhamnosidase gene TstRhaA (GenBank Accession No. ANW98449.1,CSTERTH_05025) from Thermoclostridium stercorarium subsp.thermolacticum DSM 2910 was 2787 bp in length, encoding amino acids with a pI of 5.5 and a predicted molecular mass of 106.6 kDa.TstRhaA exhibited a high homology with the α-L-rhamnosidase from Clostridium stercorarium (89.33% identity, GenBank Accession No. CAB53341.1)and Clostridiaceae bacterium (82.24% identity, GenBank Accession No. NLX78030.1).Comparison of the TstRhaA cluster with several bacterial-derived α-L-rhamnosidase of the GH78 family revealed that both possessed the same proposed general acid and base, Glu (Fig. 1) [25], suggesting that TstRhaA belongs to the bacterial members of the GH78 family.Furthermore, several charged residues (Asp442, Arg446, Asp447, Arg449, and Asp455) along the proposed general acid Glu448 were completely conserved and thus formed a conserved amino acid motif PTDCPQRDERMGWTGDA (residues 440-456).Furthermore, charged residues Arg724 along the proposed general base Glu723 were completely conserved and thus formed a conserved amino acid motif GATTIWERW (residues 717-725).The conserved amino acid motifs form the conserved catalytic domain and affect the catalytic activity of the enzyme.

Optimization of Culture Conditions
The culture conditions for the recombinant strain producing the target protein were optimized.As shown in Fig. 2A, the optimal concentration of the inducer was 0.05 mM, which indicated that 0.05 mM IPTG was the optimal concentration to induce transcription of the target gene.The optimal induction temperature was 33 o C (Fig. 2B), which showed that the speed of protein synthesis and rate by which intermediates fold into aggregates were optimal at 33 o C.Moreover, the optimal OD600 was 0.8 (Fig. 2C), indicating that the maximum balance between bacterial growth and protein expression could be achieved under this condition.Finally, the maximum enzyme activity was observed at 10 h after induction (Fig. 2D).The possible reason was that the enzyme began to be degraded by proteases after 10 h induction.Under optimal culture conditions, the target protein TstRhaA exhibited high α-L-rhamnosidase activity with approximately 25.2 U/ml, indicating that TstRhaA was highly expressed.

Purification of Recombinant TstRhaA
A Ni-TED-affinity column was used to purify TstRhaA.Finally, the yield of TstRhaA reached 16.6%, and the specific activity of purified TstRhaA was 187.9 U/mg, which was 8.0-fold higher than that of the crude enzyme (Table 1).The final obtained TstRhaA showed only one band on the SDS-PAGE gel, and its relative molecular mass was approximately 106.6 kDa, as shown by the results (Fig. 3, Lane 3).

Characterization of Recombinant TstRhaA
The enzyme properties of TstRhaA were investigated using pNPR as a substrate.As shown in Fig. 4A, the enzyme activity of TsRhaA was maximal at pH 6.5, and the enzyme activity was more than 65% of the maximum activity in a pH range of 5.5 to 7.5.The enzyme activity of TstRhaA was the highest at 65 o C (Fig. 4B), and the results showed that this enzyme was a medium-and high-temperature enzyme, similar to α-L-rhamnocidase derived from Aspergillus niger [26], Aspergillus oryzae NL-1 [19], Aspergillus terreus CCF 3059 [27] and Thermophilic strain PRI-1686 [28] (Table 2).The pH stability of TstRhaA was further determined.The results showed that more  than 94%, 90%, and 75% of the original enzyme was maintained by the residual enzyme activity of purified TstRhaA after incubation at 4°C, 45°C, and 60°C for 4 h in a pH range of 5.5 to 7.5 (Fig. 4C), respectively.However, the residual enzyme activity of α-L-rhamnosidase from Paenibacillus odorifer only retained more than 85% of the original enzyme activity after incubation at 4°C and 45°C for 1 h in the pH range of 5.5 to 7.5 [29].In addition, the residual enzyme activity of α-L-rhamnosidase from Bacteroides thetaiotaomicron also only retained more than 60% of the original enzyme activity after incubation at 45°C for 4 h in the pH range of 5.5 to 7.5 [18].Therefore, the TstRhaA has better pH stability and is suitable for use under neutral conditions.In industrial applications, the thermal stability of enzymes is a very important parameter.Therefore, the thermal stability of TstRhaA was determined at 60°C and 65°C (Fig. 4D).TstRhaA was highly stable at 60°C and 65°C, at which it maintained more than 70% of the initial activity at 10 h and more than 50% of the initial activity at 5 h.However, the residual activity of two α-L-rhamnosidase from Thermophilic bacterium PRI-1686, RhmA and RhmB, was lower than 40% and 60% after 8 h incubation at 60°C, respectively.Moreover, the half-life of the α-L-rhamnosidase from A. terreus CCF 3059 was only 127.9 min at 65°C.The residual activity of α-L-rhamnosidase from A. niger was retained at 60% after 1 h of incubation at 65°C.The results showed that higher temperatures are better when using TstRhaA, which can reduce the viscosity of the substrate, improve the solubility of the substrate, and reduce the risk of microbial contamination [19], which is beneficial for industrial applications.

Strain
Temperature * ( o C) Reference [26] * The temperature indicates optimal temperature.# Not detected, the value was not detected based on the references.
For poorly water-soluble substrates, some cosolvents are generally added to the enzymatic conversion system, such as methanol, alcohol, and dimethyl sulfoxide (DMSO).Therefore, we determined the effect of organic solvents on TstRhaA activity.As shown in Fig. 5, TstRhaA retained 50% of its remaining enzyme activity at methanol concentrations below 20%.However, the enzyme activity of TstRhaA decreased significantly when the alcohol and DMSO concentrations exceeded 15%, and the residual enzyme activity was less than 40%.The inhibition of TstRhaA by the three organic reagents was as follows: methanol > DMSO > alcohol, which was consistent with α-L-rhamnosidase from Aspergillus oryzae and Novosphingobium sp.PP1Y [19,30].These results suggest that TstRhaA can be used in reaction systems containing lower concentrations of organic solvents.

Substrate Specificity and Enzyme Kinetic Assays
A range of p-nitrophenyl-based artificial substrates were selected to test the substrate specificity of TstRhaA.TstRhaA was active against pNPR but not against pNPGlu, pNPArf, pNPArp, pNPGal, or pNPXyl.These results indicated that TstRhaA is active only against α-L-rhamnosidase.Additionally, the substrate specificity of TstRhaA was determined for four plant-derived flavonoids, including myricetrin, naringin, hesperidin, and rutin (Table 3).The results showed that TstRhaA can hydrolyze natural products containing α-1,2 and α-1,6 glycosidic bonds but not α-1 glycosidic bond natural products, which is consistent with α-L-rhamnosidase from A. oryzae [19].
Michaelis-Menten parameters were determined by enzyme kinetics studies using pNPR as a substrate at optimal temperature and pH.Table 2 shows that the K m , V max , kcat, and k kcat/K M of the enzyme were 0.36 mM, 368.3 U/mg, 650 s -1 , and 1,810 s -1 mM -1 , respectively, when pNPR was used as a substrate.The V max values were higher than those of α-L-rhamnosidase from A. oryzae, A. terreus, Thermophilic bacterium PRI-1686, and A. niger [19,[26][27][28].Furthermore, the lower K m value and higher kcat/K m values also indicate that TstRhaA exhibits high substrate affinity and catalytic activity against pNPR among the thermostable α-L-rhamnosidase.Thus, the catalytic kinetics of TstRhaA on pNPR also demonstrate that TstRhaA exhibits properties distinct from other thermostable α-L-rhamnosidase.

Analysis of Rutin Degradation
To verify that rutin undergoes biotransformation by TstRhaA, the hydrolysate was studied by HPLC.As shown in Fig. 6, the final product was identified as isoquercitrin.Furthermore, to determine the optimal conditions for the enzymatic bioconversion of rutin by TstRhaA, the conversion for isoquercitrin preparations was examined at the same substrate concentration and different temperatures, pH conditions, and enzyme dosages.As shown in Fig. 7A, the optimum temperature for hydrolyzing rutin to isoquercitrin was observed at 60 o C.This was not consistent with the results obtained by the enzymatic properties measured with pNPR as a substrate, which may  The activity against rutin was assumed to be 100%.c Not detected, relative enzyme activity is not detected.result from the thermal stability being better at 60°C than at 65°C.Moreover, the optimal pH was revealed to be 6.5 (Fig. 7B), which was identical to the result obtained by the enzymatic properties measured with pNPR as the substrate.Subsequently, the optimal enzyme dosage used to convert rutin to isoquercitrin was determined.The results showed that almost all rutin was converted to isoquercitrin in the presence of 0.3 U/ml TstRhaA in a 200 μl reaction system (Fig. 7C).To clarify the course of changes in the concentration of the two components during the reaction, a time-course experiment was carried out, and the hydrolysate was analyzed by HPLC.As shown in Fig. 7D, 60 g/l rutin was converted to 45.55 g/l isoquercitrin after 150 min of reaction.The molar conversion rate of rutin and the yield of isoquercitrin were 99.8% and 12.22 g/l/h, respectively.Compared with α-L-rhamnosidase from other sources (Table 4), TstRhaA is superior in converting high concentrations of rutin to isoquercetin.
In this work, a novel α-L-rhamnosidase from Thermoclostridium stercorarium subsp.thermolacticum DSM 2910 was overexpressed and characterized.TstRhaA displayed higher optimal temperature, better thermal  stability, and good tolerance to organic reagents, which is beneficial for application in industry.Furthermore, the enzyme exhibited high catalytic activity for the conversion of rutin to isoquercetin.Therefore, this study presents a novel α-L-rhamnosidase that could be used for the mass production of isoquercitrin.

Fig. 2 .
Fig. 2. Optimization of culture conditions of TstRhaA.(A) Effect of IPTG concentration on enzyme activity; (B) Effect of induction temperature on enzyme activity; (C) Effect of OD600 on enzyme activity; (D) Effect of induction time on enzyme activity.Data represent the means of three experiments, and error bars represent the standard deviation.

Fig. 4 .
Fig. 4. The effects of pH and temperature on the activity and stability of TstRhaA.(A) Effect of pH on TstRhaA activity.(B) Effect of temperature on TstRhaA activity.(C) The pH stability of the enzyme TstRhaA.(D) The thermostability of the enzyme TstRhaA; the residual activity was monitored, while the enzyme was incubated at 60°C (filled circles) and 65°C (filled inverted triangles).The initial activity was defined as 100%.These activities were expressed as relative values.Data represent the means of three experiments, and error bars represent the standard deviation.

Fig. 5 .
Fig. 5.The effect of organic solvents, including methanol, alcohol, and DMSO, on TstRhaA activity.The final concentration of substrate was 1.0 mM.Double distilled water was used instead of the organic solvent for the control.The average activity of the control from three experiments was defined as 100%.Others were expressed as residual activity values.Data represent the means of three experiments, and error bars represent the standard deviation.

Table 3 . Natural substrate specificity of TstRhaA.
b